P l a n t and Soil 34, 183-200 (1971)
lV~s. 1390
S T U D I E S ON T H E E F F E C T S O F C E R T A I N S A L T S ON G E R M I N A T I O N , ON G R O W T H O F R O O T , A N D ON M E T A B O L I S M I.
EFFECTS OF CHLORIDES AND SULPHATES OF SODIUM, POTASSIUM AND MAGNESIUM ON GERMINATION OF WHEAT GRAINS by A. FATHY YOUNIS and M. A. HATATA
Botany Dept., Faculty of Science, University of Alexandria, U.A.R. (Egypt)
SUMMARY I. Criteria of g e r m i n a t i o n of w h e a t grains on morphological, histological and physiological bases were studied and described to set a p a r a m e t e r for t h e assessment of g e r m i n a t i o n capacity. 2. The eifects of single salt solutions of NaC1, Na~SO4, KC1, K2SO4, lV~gC12 and MgSO4 on g e r m i n a t i o n of w h e a t grains were i n v e s t i g a t e d , and it was found t h a t : a) The g e r m i n a t i o n c a p a c i t y decreased w i t h increase of salt c o n c e n t r a t i o n b e y o n d a certain level characteristic for each salt. b) The chlorides and sulphates of each cation were equally effective on germin a t i o n at e q u i - e q u i v a l e n t concentration. c) The suppressive action of g e r m i n a t i o n decreased in t h e order Mg -+ K -+ No for e q u i - e q u i v a l e n t concentrations. 3. The suppressive action of salts oil g e r m i n a t i o n could be a t t r i b u t e d to their effect on m e t a b o l i s m and n o t to their osmotic action for t h e following reasons : a) Mannitol solutions of osmotic a c t i v i t y higher t h a n t h a t of salt solutions which p r o v e d suppressive t o g e r m i n a t i o n did n o t affect tile g e r m i n a t i o n cap a c i t y appreciably. b) R e s p i r a t i o n of m a g n e s i u m - a f f e c t e d grains was lower t h a n t h a t of control grains irt water. c) The suppressive action of t h e salts was n o t o v e r c o m e b y s u b s e q u e n t washing w i t h distilled water, especially if t h e grains s t a y e d in salt solution for 48 hours or more. I n this respect m a g n e s i u m salts were m o r e suppressive t h a n p o t a s s i u m salts. 4. The suppressive action of t h e salts on g e r m i n a t i o n could be a t t r i b u t e d to the cations r a t h e r t h a n to the anions.
184
A. FATHY YOUNIS AND M. A. HATATA INTRODUCTION
The effects of inorganic salts on plants could be related to two main processes: the osmotic processes and the metabolic processes. While it is fairly easy to eliminate the osmotic effects by using solutions of very low concentration, it is rather difficult to correlate specific salt or ion effects to specific metabolic reactions. Although the current literature reveals an increasing interest in the role of the mineral ions in metabolism as indicated by S t r o g o n o v 14, many difficulties confront the investigator in this field as pointed out by E p s t e i n 5. Further, most Studies Oil the effect of salt concentration and specific ion effects on growth deal with plants subsequent to the early seedling stage as can be derived from different reviews 1 n. The ability of a given species or variety of plant to germinate and establish the seedling is frequently the limiting factor in crop production, and under saline conditions this phase of the life cycle of the plant m a y be the critical one. The present paper deals with the effect of certain salts Oil the germination of wheat grains with the aim of finding out whether the salt effect is due to osmotic action or to specific ion effects. The rich literature on the effect of salts on germination has been reviewed b y different investigators (as for e.g. 1° 1~ 14). One of the recent studies ~ is of particular interest, for it deals with the effect of sodium Chloride on wheat grains in particular, which is the plant used in the present study. MATERIAL AND METHODS
Triticum vulgare, var. Tosson was used in this study. The grains were obt a i n e d from the D e p a r t m e n t of Horticulture, F a c u l t y of Agriculture, Univ e r s i t y of Alexandria. Two main problems confront t h e investigator in the field of germination, especially w h e n c o m p a r i n g results of different workers : the first is the fact t h a t t h e y do n o t all use the same definition for germination, and therefore t a k e different standards for their m e a s u r e m e n t s ; the second is t h a t t h e y use different techniques to test the g e r m i n a t i o n capacity. This feature has been p o i n t e d out b y reviewers of the subject (as for e.g. 10). Concerning t h e first problem, the ' B o t a n i s t ' s definition' of germination, n a m e l y the emergence of radicle from seed coat, was acknowledged for t h e present study. I t was ascertained b y morphological and histological investi-
pl.
rad. A. 8 hours
col. end.
pl.
rad. col. B. 16 hours
end. col.
Plate I. A selection of successive stages of germinating wheat grains. Photos a, b and c are close-up views of whole grains as seen from the grooved, the convex and the lateral sides respectively. Photo d is a photomicrograph of a longitudinal section cut through the embryo. The scales on series d represent 1 millimeter. Abbreviations: pl = plumule, tad = radicle, col ~ coleorhiza, end = endosperm.
tad. co]-.
C. 24 hours
end. col.
col.
pl.
col. root
D . 32 h o u r s
P l a t e 2.
See P l a t e 1.
EFFECTS OF SALTS ON GERMINATION AND GROWTH
185
gation of the embryo; and was endorsed further by following the course of carbon dioxide output during germination after soaking the grains in water. As to the second problem, a series of experiments was performed using the petri dish method commonly employed, in which the dry grains were spread over a pad of filter paper in the dish, and few millimeters of water or solution added to cover the grains. Results of the experiments in which this procedure was followed were rather inconsistent and irregular. On close investigation it was found that if the grain happened to rest on the wet filter paper with the embryo (converse side) touching the filter paper, it usually failed to germinate in the effective salt solution; and sometimes even in water. If the grain happened to rest with the grooved side on the wet filter paper the embryo was then raised above the level of the solution and it usually germinated even in the high concentration of salt solution. Increasing the level of the solution on the filter paper to cover the grains was tried out in order to overcome this difficulty, the result was still worse; for even with grains in water in this manner the per cent of germination was very low. This was probably due to inadequate oxygen supply to the embryo. The procedure which proved to be more satisfactory and gave very consistent and reproducible results was as follows : fifty grains were placed in a 200-ml conical flask containing 150 mt water or salt solution. Each flask was stoppered and provided with inlet and outlet delivery tube for aeration. All experiments were conducted in the dark at 26°C in a thermostatically controlled incubator. Further details are given afterwards in due place. EXPERIMENTS AND RESULTS
Experiment z: Morphological and histological studies o/wheat grains germinated in water Eight batches of 20 selected wheat grains were washed thoroughly and germinated in continuously aerated distilled water at 26°C. One batch of the grains was recovered every 4 hours for 32 hours, examined carefully and a representative sample was photographed with close-up lens. All grains were dropped in a weak chrome-acetic fixer, dehydrated, embedded in wax, and cut longitudinally accross the embryo according to standard histological techniques. The sections were stained in saffranin-fast green according to a procedure modified after a schedule described by J e n s e n 9 P l a t e s 1 a n d 2 s h o w p h o t o s r e p r e s e n t i n g s u c c e s s i v e s t a g e s of t h e g e r m i n a t i n g w h e a t g r a i n s a t 8, 16, 24 a n d 32 h o u r s f r o m s o a k i n g . T h e s t u d y of t h e g e r m i n a t i n g w h e a t g r a i n s r e v e a l e d t h a t t h e first v i s i b l e s w e l l i n g of t h e e m b r y o a p p e a r e d a f t e r 8 h o u r s of s o a k i n g , b u t t h e s w e l l i n g w a s n o t m a r k e d b y a n y g r o w t h o u t of t h e g r a i n ' s p e r i carp. A f t e r 16 h o u r s s o a k i n g a d i s t i n c t w h i t e s t r u c t u r e a p p e a r e d o u t of t h e g r a i n , it is t h e c o l e o r h i z a w h i c h w h e n v e r y s m a l l m a y b e e a s i l y
186
A. F A T H Y Y O U N I S AND M. A. HATATA
m i s t a k e n for a first root. T h e coleorhiza enlarged a n d swelled u p considerably after 16 hours soaking, a n d this m o r p h o l o g i c a l change was a c c o m p a n i e d w i t h increasing cell division a n d differentiation in the root's m e r i s t e m . After 28 hours soaking w h e n the tissues of the first root were well defined, this root s t a r t e d to pierce its w a y out of the coleorhiza. T h e s t a t e of the seedling after 32 h o u r s (Plate 2) could therefore be r e g a r d e d as the p r o p e r g e r m i n a t i o n according to the B o t a n i s t ' s definition. I n all s u b s e q u e n t studies of g e r m i n a t i o n of w h e a t positive g e r m i n a t i o n was a c k n o w l e d g e d at the a t t a i n m e n t of a stage similar to t h a t shown b y the p h o t o t a k e n a f t e r 32 hours soaking in distilled water. T h e first root in such a case was only 2 to 3 m m long.
Experiment 2." Course o/COs output o/germinating wheat grains Tell grams of selected wheat grains were washed thoroughly and placed in a gas-wash bottle provided with sintered glass bubbler at the inlet tube; 75 ml distilled water were then added to the grains. CO2-free air was bubbled through the soaked grains and the outflowing air was passed in a second gaswash bottle containing 75 ml standard Ba(OH)s solution to absorb the COs evolving from the grains. A blank control was run simultaneously alongside with an identical set-up, but without grains. Three replicates with grains and two blanks were run at a time. The experiment was conducted at 26°C in the dark. At regular 2-hours intervals for 6 hours, then at one-hour interval for 30 more hours the Ba(OH)2 was titrated against standard 0.02 N HC1 using phenolphthalein and stirring the solution with COs-free air. The rate of COs output of the grains was calculated in terms of mg COs per 100 g air-dry grains per hour, after making allowance for the values of the blank control. T h e results of this e x p e r i m e n t are given in Figure 1, I ; t h e y are a v e r a g e values of three replicates. T h e figure shows also a second c u r v e m a r k e d I I for the values of C02 o u t p u t f r o m grains soaked in MgS04 solution; the i n t e r p r e t a t i o n of this c u r v e will follow l a t e r (refer to e x p e r i m e n t 8, p. 188). The results of this e x p e r i m e n t show t h a t the r a t e of respiration of w h e a t grains r e m a i n e d r a t h e r low, not exceeding 6 mg/100 g grains/ h o u r for the first six hours following soaking; this period is m a r k e d A on the curve. After t h a t the r a t e of respiration increased s h a r p l y to 14 m g a f t e r 7 hours, t h e n to 19.4 m g after 8 hours. T h e 8th h o u r seems to be the m o m e n t of the o u t b u r s t of respiration. F r o m the 8th to the 26th h o u r after soaking the r a t e of respiration was s o m e w h a t s t e a d y at a p p r o x i m a t e l y 22 m g / h o u r ; this period is m a r k e d B on t h e
EFFECTS OF SALTS ON GERMINATION AND GROWTH
DtsliHedw;ter I
~'~ 32
187
I
28
~.
2~ 20
Mgso so mo/L
12
n
8
8
12
16
20
24
28
32
3G hours
tepse ef time efter soeklng
Fig. 1. Rates of carbon dioxide output of wheat grains germinated in: I Distilled water, and II MgSO4 solution of 50 me/1 Stage A coincides with the beginning of swelling of coleorhiza; stage B with m a x i m u m swelling of coleorhiza; stage C with the rupturing of coleorhiza and emergence of root.
curve. After 28 hours from soaking a further rise in the rate of respiration was noticed, this period is marked C on the curve. It is particularly interesting to correlate these physiological activities with the morphological and histological changes observed in the grains. The change from A to B could be associated with the beginning of the swelling of the embryonic region as a result of the expansion of the coleorhiza. At this stage no vacuoles were noticeable in the meristematic cells of the radicle. Stage B could be associated with the steady development of the radicle and the rapid expansion and protrusion of the coleorhiza. At the 12th hour very small vacuoles were evident in the pro-epidermal and pro-cortical meristems of the radicles; and at the 16th hour large vacuoles were evident in all cells except in the region of the plerome in which the vacuoles appeared at the 20th hour. Stage C could be associated with the appearance of the radicle and coleoptile outside the pericarp and coleorhiza; all the cells of the radicle except the very young primordia were clearly vacuolated by that time. The changes in the course of respiration of germinating wheat grains endorse the conclusion arrived at from the study of the course of morphological and histological changes, namely that positive germination could be acknowledged when the radicle pierces the coleorhiza which is also the moment of change in respiration rate from level B to a higher level C as shown b y curve I of Figure 1.
188
A. F A T H Y Y O U N I S AND M. A. HATATA
Experiments 3 to 8: E//ect o~ diHerent concentrations o~ sodium potassium or magnesium chloride or sulphate on the germination o! wheat grains Using the conical flak procedure described above, the effect of a series of single salt solutions of Na, K or Mg chloride or sulphate of the following concentrations : 0, 1, 5, 10, 20, 30, 40, 50, 100 and 200 milliequivalent per litre (me/1 thereafter) was investigated; every concentration was run in triplicate. The solution was renewed every 24 hours when its pH was measured and the grains examined to spot and pick out those which showed positive germination according to the standard set above. The experiments were carried out at 26°C ill the dark, and continued for 5 days when it became evident that no more grains would germinate. The osmotic value of every solution was determined cryoscopically. The average values of germination per cent, and the standard error of the mean of these values were calculated.
T h e results are given in tile collective Figure 2. T h e figure shows t h a t in distilled w a t e r no g e r m i n a t i o n was noticed after 24 hours f r o m soaking; b u t after 48 hours the per cent of g e r m i n a t i o n was 90 a n d c o n t i n u e d to increase slightly a f t e r t h a t to a m a x i m u m value of 93 per cent. Generally s p e a k i n g it is evident f r o m the figure t h a t there was no significant difference b e t w e e n the effect of the chloride a n d s u l p h a t e of e v e r y p a i r of salts at e q u i - e q u i v a l e n t concentration. I t should be noted, however, t h a t the chlorides h a v e higher osmotic activities t h a n the s u l p h a t e s of equi-equivalent concentration. I t is also evident t h a t the g e r m i n a t i o n c a p a c i t y decreased w i t h increasing salt c o n c e n t r a t i o n in all of the six different salt species. H o w e v e r , t h e r e are m a r k e d specific differences associated with salt effects : the c o n c e n t r a t i o n of the single salt solution which s u p p r e s s e d g e r m i n a t i o n per cent to one a n d the s a m e e x t e n t decreased in the order s o d i u m - - > p o t a s s i u m - > m a g n e s i u m . This m e a n s t h a t m a g nesium in association w i t h chloride or s u l p h a t e s u p p r e s s e d germin a t i o n of w h e a t m o r e s t r o n g l y t h a n corresponding p o t a s s i u m or sodium salts. T h e correlation b e t w e e n osmotic a c t i v i t y a n d t y p e of salt is described later, after E x p e r i m e n t 9. W h e n it b e c a m e so clearly evident t h a t m a g n e s i u m salts suppressed the g e r m i n a t i o n of w h e a t strongly, it was felt desirable to correlate this suppression w i t h the m e t a b o l i c a c t i v i t y of the e m b r y o in a simple m a n n e r b y following the course of C02 o u t p u t in the presence of m a g n e s i u m salts. F o r this p u r p o s e w h e a t grains were soaked
E F F E C T S OF SALTS ON G E R M I N A T I O N A N D G R O W T H
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190
A. FATHY YOUNIS AND M. A. HATATA
in a 50 me/1 magnesium sulphate solution (which was found to suppress germination definitely within 48 hours by 70 per cent from normal) and the course of CO.) output was followed continuously at short intervals for 36 hours as described above under Experiment 2. The results are given in Figure 1, II. It is interesting to notice from this figure that the grains soaked in magnesium sulphate continued to respire quite normally for 20 hours; they even showed the sharp increase in CO2 output after 8 hours of soaking, exactly as did the grains soaked in water. However, after 20 hours in the magnesium sulphate solution the rate of CO2 output began to drop down to approximately 50 per cent of the normal rate. It was shown in Experiment 1 that the interval of 20 to 24 hours from the start of soaking in water was marked by the swelling of the coleorhiza and the active development of the radicle, and the differentiation of its tissues. It may be concluded, therefore, that the suppressive action of magnesium sulphate must be due to an inhibitive action of magnesium ions on some metabolic process associated with differentiation and development of the embryo.
Experiment 9: The role o/osmotic activity on germinalion A study of the effect of different concentrations of mannitol on the germination of wheat grains was carried out to investigate the role of the osmotic activity of a saltless solution on germination. The procedure was exactly the same as that used in experiments 3 to 8 above, and the concentrations of mannitol used were 20, 35, 50, 75 and 125 g/litre, which furnished a range of osmotic activity extending between 2.9 and 12 atmospheres. The results of this experiment are given in Figure 3. It is obvious from the results of this experiment that an increase in the osmotic activity of the bathing solution to 2.9 atmospheres did not reduce the germination capacity of wheat by any appreciable value; the reduction was 2 per cent only. With increasing osmotic activity there was an increased suppression of germination. A 10 per cent suppression was noticed with mannitol solution containing 50 to 70 g/1 which has an asmotic activity of 7.2 to 9.6 atmospheres. The highest concentration of mannitol used namely 125 g/1 which has an osmotic activity of 12 atmospheres suppressed germination by 30 per cent. For an easy comparison between the effects of the different salt species and their osmotic activities on the germination capacity of
EFFECTS
OF SALTS ON GERMINATION
Expl. 9
AND GROWTH
191
Mannilo[
90 S01 E E
60 60
"~
~0
ra_ 3O
24 46 22 96 120 hours Lopse of lime error seeking
Fig. 3. Effect of different concentrations of mannitol on the germination capacity of wheat grains. The values are means of three replicates of 50 grains each. The numbers printed against every curve indicate the osmotic pressure of the solution in atmospheres; D.W. = distilled water. The broken lines accross the curve represent the levels of suppression of germination by 10 and S0 per cent from control in water. w h e a t grains a s u m m a r y of the m a i n results of E x p e r i m e n t s 2 to 9 is p r e s e n t e d in T a b l e 1. I f one considers a 10 per cent suppression of g e r m i n a t i o n as a significant effect of salt action, a n d 50 per cent suppression as r a t h e r serious action, t h e n b y reference to T a b l e 1 it could be e a s y to c o m p a r e b e t w e e n the effects of the different salt or m a n n i t o l c o n c e n t r a t i o n , a n d to correlate these effects with the osm o t i c a c t i v i t y of the b a t h i n g solution. I t is evident f r o m the table t h a t t h e 50 per cent suppression was induced b y 30 me/1 MgC12 or MgS04; 50 to 100 me/1 KC1 or K2S04, 200 me/1 NaC1 or Na2S04. T h e higher s u p p r e s s i v e action of m a g n e s i u m salts o v e r p o t a s s i u m salts, a n d these o v e r s o d i u m salts could not be a t t r i b u t e d to osmotic phen o m e n a since, as it is s h o w n in T a b l e 1, the osmotic a c t i v i t y of the solutions of these salts were m u c h lower in case of m a g n e s i u m t h a n in p o t a s s i u m t h a n in s o d i u m ; a n d t h e y were all lower t h a n the act i v i t y of m a n n i t o l . I t m a y be concluded, therefore, t h a t in their chloride or s u l p h a t e form, m a g n e s i u m is m o r e toxic t h a n p o t a s s i u m which in t u r n is m o r e toxic t h a n sodium salts of e q u i v a l e n t concentration, a n d t h a t the t o x i c i t y is due to specific ion effect not to osmotic phenomena.
192
A. FATHY YOUNIS AND M. A. HATATA TABLE 1
A comparison between the effective salt or mannitol concentrations which suppressed the germination capacity of wheat grains by 10 per cent and by 50 per cent from the normal germination capacity in distilled water within 120 hours. Values derived from the results of experiments 3 to 9 Solutions which suppressed germination by 10 per cent Solute
NaC1 NasS04 KCI KzSO4 MgC12 MgSO4 Mannitol
Concentration in me]l
g]l
Osmotic pressure atm.
40 40-50 20-30 20 10 10-20 --
2.3 2.8-3.5 1.5-2.2 1.7 1.0 1.2-2.4 50-70
2.04 1.3 -1.7 0.96-1.3 0.66 0.48 0.24-0.5 7.2 -9.6
Solutions which suppressed germination by 50 per cent Concentration in me]l
g]l
Osmotic pressure atm.
200 200 50-100 50-100 30 30 --
11.7 14.2 3.7-7.4 4.3-8.7 3.0 3.6 > 100
9.7 6.0 2.4-4.68 1.7-3.70 0.96 0.60 > 12
Experiments zo to z3: The extent o/the suppressive action o/chlorides and sulphates o/potassium and magnesium on the germination capacity o/wheat grains Since t h e s u p p r e s s i v e a c t i o n of t h e s e c a t i o n s b e c a m e e v i d e n t m o r p h o l o g i c a l ly w i t h i n 48 h o u r s a n d i n c r e a s e d t o its m a x i m u m w i t h i n 72 hours, i t w a s t h o u g h t w o r t h w h i l e t o i n v e s t i g a t e t h e e x t e n t of s a l t t o x i c i t y o n t h e e m b r y o , a n d w h e t h e r t h e t o x i c i t y was r e v e r s i b l e or n o t b y t e s t i n g t h e v i a b i l i t y of t h e g r a i n s following s a l t b a t h i n g for d i f f e r e n t t i m e periods. F o r t h i s p u r p o s e 50 w h e a t g r a i n s were s o a k e d in t h e desired single s a l t s o l u t i o n for 24, 48 or 72 h o u r s w i t h c o n t i n u o u s a e r a t i o n . T h e g r a i n s were t h e n w a s h e d t h o r o u g h l y w i t h distilled w a t e r , a n d t h e i r g e r m i n a t i o n c a p a c i t y in distilled w a t e r was t h e n i n v e s t i g a t e d o v e r a p e r i o d of t i m e (usually 3 days) long e n o u g h t o secure t h e a t t a i n m e n t of m a x i m u m g e r m i n a t i o n . T h e s o l u t i o n s or w a t e r were r e n e w e d e v e r y d a y w h e n t h e g r a i n s were e x a m i n e d t o p i c k o u t t h o s e w h i c h s h o w e d p o s i t i v e g e r m i n a t i o n a c c o r d i n g t o t h e s t a n d a r d set before ( e x p e r i m e n t 1 above). T h e s t u d y was c o n f i n e d t o t h o s e p o t a s s i u m or m a g n e s i u m s a l t c o n c e n t r a t i o n s w h i c h p r o v e d effective in r e d u c i n g t h e g e r m i n a t i o n c a p a c i t y of w h e a t grains. T h r e e r e p l i c a t e s of e a c h c o n c e n t r a t i o n a n d t w o sets of c o n t r o l were r u n s i m u l t a n e o u s l y ; in t h e f i r s t c o n t r o l set t h e g r a i n s were s o a k e d c o n t i n u o u s l y in distilled water, a n d ill t h e second c o n t r o l t h e g r a i n s were s o a k e d c o n t i n u o u s l y i n t h e single s a l t s o l u t i o n u n d e r i n v e s t i g a t i o n . T h e r e s u l t s of t h i s e x p e r i m e n t
a r e g i v e n i n t h e c o l l e c t i v e F i g u r e 4.
The figure shows that the maximum
germination
p e r c e n t of w h e a t
193
EFFECTS OF SALTS ON GERMINATION AND GROWTH
Expf. 11
Expt. 10 lO0
Expt. 12
~ so so ,~l/L
KCI 50 ml/L
100
9C
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40 36
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C Hours in MgU20 24 48 72 120 0 24 48 72 120 Hours in D.W 120 72 ?2 ?2 0 120 72 F2 72 0 Treatment in hours
Mg S~ 50 m//L
90 80
[] in w a t e r ~in solt
"s 'd ¢t.
Hours in Mg SoO 24 48 72 120 Hours in D.~ 120 ?2 ?2 72 0
24 48 72 120 120 72 72 72 0
Treatment in hours
Fig. 4. G e r m i n a t i o n c a p a c i t y of w h e a t grains soaked in 30 or 50 me/1 of chlorides or sulphates of p o t a s s i u m or m a g n e s i u m for different d u r a t i o n s followed b y washing in w a t e r for 72 hours.
grains in distilled water was 94. Soaking the grains in 50 me/1 KC1, or K2S04 or 30 and 50 me/1 MgC12 or MgS04 for 24 hours only, then washing the grains in distilled water for three days reduced the maximum germination per cent by approx. 20. When the soaking period in salt solutions was extended to 48 hours followed by washing in
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distilled water for 3 days, the maximum germination per cent dropped down to about 50 in potassium salts and to about 40 in the magnesium salts; these values were slightly higher than the values of germination when the grains were soaked continuously in the salt solution without subsequent washing. The effect was more pronounced still when the soaking period was extended to 72 hours and followed by 72 hours washing in distilled water. These results indicate that the suppression of germination by these concentrations of potassium or magnesium salts were rather permanent and not removeable b y subsequent washing, and must be regarded as a feature of salt toxicity. The toxic action is also associated with ion specificity since the effect of magnesium salts was more pronounced than that of equiequivalent potassium salts. Further, it m a y be concluded that the effect of these salts Could not be due to osmotic activity because : a) the suppression was not eliminated by washing with distilled water, and b) in mannitol solution of osmotic pressure 2.9 atmospheres (Exp. 9 above) the germination capacity of wheat was not reduced at all; the osmotic activity of this mannitol solution is much higher than the osmotic activity of the salt solutions which reduced germination per cent irreversibly. In all cases recorded in this series of experiments (10 to 13), including those in which the suppressive action was irreversible in respect to the first seminal root, it was noticed that when the grains were transferred from the salt solution to distilled water a certain degree of activity was initiated in the embryo. The coleoptile together with few adventitious roots developed out of the grains quite normally following the washing out of the salt from the medium. This feature indicates that the salt toxicity was exerted upon that part of the embryo - the first seminal root - that happened to start its activity in the presence of the salt; and the quescent tissues were not harmed b y the salt. Furthermore, since there was no significant difference between the effects of chlorides and sulphates of equiequivalent concentrations in association with K or Mg, whereas the Mg salts were always more toxic than the corresponding K salts, it must be concluded that the toxicity is due to the action of the cations rather than t h a t of the anions.
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DISCUSSION
The importance of the results of the experiments described above could be realized from the fact that organized studies in this field date back to 1898, and a massive literature is now available; but most of these studies deal with the problem of the overall salinity and its effect on germination. The early studies have been reviewed by different authors 6 11 16 who agreed on that germination of seeds is retarded by salts in solutions, and that the degree of delayed germination and injury of seeds and seedlings is in direct proportion to the osmotic pressure of the saline solution. Some of these early investigators however 6 7 13 16 have pointed out the specific ion effects on germination and development of the embryo. More recent studies in the field of salt effects on germination and growth have been reviewed by different workers 2 10 14 15. In general, the different investigators agree on that the salt effects on germination are rather specific to salt or ion species on one hand, and to plant species on the other hand. The studies with single adsorbed ions 4 are of particular significance in this field. In the review of the works of Russian and other scientists in this field it was shown (Ref. 14, p. 22) that for the germination of seeds not only the concentration but also the ionic composition of the solution is important; and that the detrimental effect of ions on the growth of embryos of Triticum, Avena, Secale and other plants apparently follows the same order as the arrangement of the ions in the lyotropic series, i.e. CNS > NO3 > C1 > Br > H2P04 > S04; and for the cations Li > NH4 > Ba > Ca > K > Na > Mg. Nevertheless, m a n y of the studies presented in that review 14 lead to the conclusion that the suppression of germination by salts is a consequence of the osmotic activity, which is contrary to the results of the experiments described in the present paper. It is rather important at this stage to draw attention to the fact that while some investigators examined the effect of salts on germination using single salt solution, others used balanced nutrients with the extra addition of the desired salt sPecies, and others still added salt solutions to seeds in the soil. Such variations in the procedure add to the complexity of the problem, for as it has been pointed out before s the evaluation of the level of salinity conditioning the germination of seeds under field conditions is difficult, since the amount of soil moisture and the
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salt concentration are changing continuously owing to evaporation, capillary transmission of water, and rainfall or irrigation. Further, the use of balanced solution with an extra addition of salts offer some difficulties in the interpretations because of the antagonism and synergism between component ions. The same objection has been raised before (Ref. 14, p. 21) when it was stated that a balanced solution is less favourable for inhibition than either pure NaC1 or Na2S04 since, because of ion antagonism, fewer sodium ions are absorbed from a balanced solution than from pure solutions of NaC1 or Na2S04. From this discussion it seems more appropriate, therefore, to use single salt solutions for the study of salt effects on germination and to varify the osmotic action by conducting a check up experiment using non-salt solutions as mannitol, which was carefully done in the experiments described above. The main features of the results of this study are: a) The germination of wheat grains was suppressed by chlorides and sulphates of Na, K or Mg to the same extent in respect to the anion, indicating a cation effect rather than an anion effect. In this connection reference should be made to a previously published remark (Ref) 4, p. 23) that 'the results of m a n y experiments using isoosmotic solutions showed NaC1 to be less harmful than Nag.S04 to m a n y plants. Therefore, the widespread view that chlorides are more toxic than sulphates is not always correct'. b) The order of effectiveness in suppression of germination of wheat was Mg > K > Na. c) The suppression was not due to osmotic activity as it was not induced by mannitol solutions of osmotic activity equal to or even higher than that of the salt solution. d) The suppressive action was irreversible and unwashable if the seeds remained in salt solution for 48 hours or more. It is rather important at this stage to refer to a recent study 3 in which the effects of NaCl and CaCI2 on the germination of wheat grains have been investigated, since the results reported in that work disagree to some extent with the results of the experiments described in the present paper. According to their figures the per cent of germination was 94 in water, 90 in I per cent NaCl and 14 in 2 per cent NaCl. B y simple calculations it is found that a one per cent NaCl
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corresponds to 170 me/1. From the results of experiment 3 above it could be seen that a 100 me/1 NaC1 suppressed the germination capacity of wheat to 57 per cent, and a 200 me/1 solution suppressed it to 30 per cent of normal control in water. Accordingly a one per cent NaC1 solution should suppress the germination capacity to a value lying between 57 and 30 per cent which is much higher than the value reported by C h a u d h u r i and W i e b e 3 namely 10 per cent only. The discrepancy m a y be attributed to: a. Difference in response of wheat varieties used. b. Difference in techniques, since C h a u d h u r i and W i e b e used the petri dish technique which gave inconsistent results in our study for the reasons stated above (p. 185). c. C h a u d h u r i and W i e b e washed the grains after 6 hours soaking in salt solution; while in our study the grains were left continuously in the salt solution for 120 hours. Attempts to correlate the suppressive action of salts on germination with their effect on metabolism did not receive adequate attention. This could be noticed from the studies of S t r o g o n o v (Ref. 14, p. 10 and 22). A careful analytical study of the uptake of water and CO~ production which take place during the germination of barley grains has been described 2, but no detailed study of metabolism is reported in that review. A modest attempt to analyse the stages of germination and their relation to metabolism through the study of C02 output has been made above (Expt. 2). This experiment showed that the suppressive action of magnesium sulphate on germination was accompanied by a decrease in the rate of respiration. It seems very desirable, therefore, to expand the study in this field. As to the extent of the suppressive action of salts on germination, it has been quoted (Ref. 14, p. 21-22) that from a study of the imbibition and salt content of cotton seeds under conditions of high salinity, it was found that the higher the soil salinity the higher t h e chloride content of the nongerminated seeds, while no difference in the water content of the seeds was noticed. Such nongerminating seeds were rinsed thoroughly and half were used for chloride determination while the remainder were sown in nonsaline soil. Despite the sharp decrease in chloride content after rinsing, and despite the absence of salts in the soil, the seeds failed to germinate. Therefore, the high concentrations of salt adversly affected the seeds during
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imbibition and as a result they lost their viability. It was also quoted (Ref. 14, p. 17) that the osmotic pressure of soil solutions up to 4.3 atmospheres do not exert any harmful effects on plants, between 4.3 and 5.2 atmospheres the growth of plants is retarded, and above 8.3 atmospheres seeds do not germinate. These two quotations reflect two opposite views: the former quotation favours a view that salts affect germination and growth through their specific ionic action; while the latter quotation favours the view that salts affect germination and growth as a result of their osmotic action. These opposite views have been echoed repeatedly in the literature. Experiments 10 to 13 described above clearly support the view that the salts examined affect germination through their specific ions and not through their osmotic activity. Nevertheless, it is worthwhile to try to find out the reason for such disagreement on salt effect on germination. One of the main reasons for this lies in the fact that some investigators acknowledge germination when the aerial organs appear above the soil (user's definition), while others abide to the Botanist's definition of germination which is acknowledged b y the emergence of the radicle ( L a n g s). The importance of selecting a parameter for assessing germination could be realised from the remark stated above (p. 194) namely that the solutions of magnesium or potassium salts which suppressed the emergence of the first seminal root did not suppress the emergence of the coleoptile or adventitious roots, especially after washing the salt-affected grains with water. This means that from one and the same experiment one m a y conclude that the salt suppressed germination - i n terms of a Botanist's definition - or that it did not suppress germination but delayed emergence if one follows the user's definition. In the light of this discussion the conclusions drawn b y M a n d y and P a l 12 that 0.1 to 0.25 per cent NaC1 stimulated germination of sunflower and wheat, and up to 3 per cent NaC1 increased germination speed in rye, i.e. reduced emergence time, m a y have a special significance. In terms of milliequivalent, 0.1 per cent NaC1 = 20 me/1 0.25 per cent = 45 me/l, and 3 per cent = > 500 me/1. From the results of experiments 3 and 4 described above it was shown that a solution of 200 me/1 NaC1 suppressed germination of wheat grains by more than 50 per cent. The 3 per cent NaC1 of M a n d y and P a l would, therefore, induce far greater suppression of germination. The
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fact that M a n d y and P al were interested in emergence rather t h a n germination in the botanical sense m a y account for the disagreem e n t in this case as well as in other similar cases. The disagreement between the results of C h a u d h u r i and W i e b e a and those recorded in the present paper concerning the effect of sodium chloride on germination of wheat m a y also be attributed to difference in acknowledging the state of germination. For, while in our study germination was acknowledged b y the emergence of the first seminal root - a feature substantiated b y the s t u d y of the change in respiration activity and histological criteria - C h a u d h u r i and W i e b e acknowledged germination w h e n three rootlets, the largest at least 5 m m long, had emerged from the grain within 3 days. A point which deserves further investigation is the c o m m o n l y noticed feature t h a t the suppressive action of salts on seeds is not a case of all or non effect. Should this be taken to mean that within the same batch oI seeds or grains one m a y have various degrees of salt tolerance ? Received October 2, 1969. Revised May 1970
REFERENCES 1 2 3 4 5 6 7 8 9 10 11
B e r n s t e i n , L., and H a y w a r d , H. E., Physiology of salt tolerance. Ann. Rev. Plant Physiol. 9, 25-46 (1953). B r o w n , R., Physiology of seed germination. Encyelop. Plant Physiol. R u h l a n d , W. (ed). XV, 894-924 (1965). C h a u d h u r i , I. I., and W i e b e , H. H., Influence of calcium pretreatment on w h e a t germination on saline media. Plant and Soil 28, 208-216 (1968). E l g a b a l y , M. M., Specific effects of adsorbed ions on plant growth. Soil Sci. 80, 235-248 (1955). E p s t e i n , E., Mineral metabolism, ia Plant Biochemistry, B o n n e t , J., and V a r n e r , J. E. (eds). Academic Press (1965). H a r r i s , F. S., Effect of alkali salts in soils on the germination and growth o5 crops. J. Agr. Research 6, 1-53 (1915). H a r r i s , H. S., and P i t t m a n , D. W., Relative resistance of various crops to alkali. U t a h Agr. Expt. Sta. Bull. 168 (1919). H a y w a r d , H. E., Plant growth under saline conditions. In: Reviews of research on problems of utilization of saline water. UNESCO, April 1954. J e n s e n , W. A., Botanical Histoehemistry. Freeman & Co (1962). L a n g , A., Effects of some internal and external conditions on seed germination. Eneyelop. Plant Physiol. R u h l a n d , W. (ed). XV, 848-893 (1965). M a g i s t a d , O. C., PIant growth relation on saline and alkali soils. Botan. Rev. 2, 181-230 (1945).
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13 14 15 16
E F F E C T S OF SALTS ON G E R M I N A T I O N AND GROWTH M a n d y , G., and Pal, G., The effect of treatment with different salt solution on seed germination in some varieties of rye, oats and sunflower. NSvenytermele (Budab.) 9, 343-358 (1960). Quoted by L a n g , A. 1965. R u d o l f s, W., Influence of water and salt solution upon absorption and germination of seeds. Soil Sci. 20, 15-37 (1925). S t r o g o n o v , B. P., Physiological Basis of Salt Tolerance of Plants. (English translation from Russian) (1964). T o o l e , E . H . , H e n d r i e k s , S . B . , B a r t h w i e k , H . A . , a n d T o o l e , E. K.,Physiology of seed germination. Ann. Rev. Plant Physiol. 7, 299-324 (1956). U h v i t s , R., Effect of osmotic pressure on water absorption and germination of alfalafa seeds. Am. J. Botany 33, 278-284 (1946).